DOI:http://dx.doi.org/10.7314/APJCP.2015.16.13.5191 Arsenic Trioxide Promotes Paclitaxel Cytotoxicity in Resistant Breast Cancer Cells

RESEARCH ARTICLE

Arsenic Trioxide Promotes Paclitaxel Cytotoxicity in Resistant Breast Cancer Cells Tayebeh Oghabi Bakhshaiesh1,2, Marzie Armat1,2, Dariush Shanehbandi3, Simin Sharifi4, Behzad Baradaran3, Mohammad Saeed Hejazi1,4*, Nasser Samadi1,5,6*

Abstract

A partial response or resistance to chemotherapeutic agents is considered as a main obstacle in treatment of patients with cancer, including breast cancer. Refining taxane-based treatment procedures using adjuvant or combination treatment is a novel strategy to increase the efficiency of chemotherapy. PPM1D is a molecule activated by reactive oxygen species. whose expression is reported to modulate the recruitment of DNA repair molecules. In this study we examined the impact of arsenic trioxide on efficacy of paclitaxel-induced apoptosis in paclitaxel-resistant MCF-7 cells. We also investigated the expression of PPM1D and TP53 genes in response to this combination treatment. Resistant cells were developed from the parent MCF-7 cell line by applying increasing concentrations of paclitaxel. MTT assays were applied to determine the rate of cell survival. DAPI staining using fluorescent microscopy was employed to study apoptotic bodies. Real-time RT-PCR analysis was also applied to determine PPM1D mRNA levels. Our results revealed that combination of arsenic trioxide and paclitaxel elevates the efficacy of the latter in induction of apoptosis in MCF-7/PAC resistant cells. Applying arsenic trioxide also caused significant decreases in PPM1D mRNA levels (p<0.05). Our findings suggest that arsenic trioxide increases paclitaxel-induced apoptosis by down regulation of PPM1D expression. PPM1D dependent signaling can be considered as a novel target to improve the efficacy of chemotherapeutic agents in resistant breast cancer cells.

Keywords: Combination therapy - arsenic trioxide - resistant breast cancer - taxanes - Wip1

Asian Pac J Cancer Prev, 16 (13), 5191-5197

Introduction that receive paclitaxel as the first or secondary-line treatment, have not responded completely to the agent. Breast cancer, the most common cancer among Different molecular mechanisms can be complicated the women, is the second cause of death after lung cancer, with taxol resistance, including changes in metabolism of the approximately 40,000 occurrence in American females chemotherapuetic agents (Kavallaris, 1997), modifications (Siegel et al., 2013). Despite marked improvement in in the dynamics of microtubules (Goncalves et al., 2001), diagnosis and long term disease-free survival because of over expression of multidrug resistance gene products improvement in current treatment; the high rate of death (Gottesman, 2002) and alteration in expression of genes, extremely caused by the resistance to chemotherapy (Zekri which are participated in cellular responses to DNA et al., 2013; Sabzichi et al., 2014). damage like PPM1D (Bauer et al., 2010). Paclitaxel is an anti-mitotic agent which is usually PPM1D was identified as the protein phosphatase, used against a wide range of solid tumors include locally which has a p53-dependent expression in response to advanced and metastatic breast cancer (Bauer et al., 2010). DNA-damage (Fiscella et al., 1997; Rossi et al., 2008). Paclitaxel as a microtubule-stabilizing agent induces It regulates several different networks in the cell cycle by apoptosis in cancer cells by activation of the mitotic check selectively inactivating some central tumor suppressor points and subsequent mitotic blockage, which is resulted pathways (Takekawa et al., 2000; Lu et al., 2008). This in inhibition of cancer cell proliferation (Burns et al., 2003; phosphatase has main activity in destruction of DNA Herbst and Khuri, 2003; Ikui et al., 2005). Studies showed damage-induced apoptosis (Ali et al., 2012). It has been that paclitaxel can induce DNA single strand breaks too shown that PPM1D as a growth-promoting phosphatase (Branham et al., 2004). plays a crucial role in tumor progression, poor prognosis As the acquired resistance increased; many patients and resistance to various chemotherapeutic agents,

1Drug Applied Research Centre,2Students’ Research Committee, 3Immunology Research Center, 4Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, 5Department of Biotechnology, Faculty of Advanced Medical Sciences, 6Department of Biochemistry and Medical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran *For correspondence: [email protected] ; [email protected] Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 5191 Tayebeh Oghabi Bakhshaiesh et al including doxorubicin and cisplatin (Ali et al., 2012; Rivas from CinnaGen (Tehran, Iran) and Central Institute of et al., 2012; Wang et al., 2012). Epidemiology (Moscow, Russia), respectively. Power Arsenic trioxide, one form of arsenicals, has approved SYBR®Green PCR Master Mix was obtained from as anticancer components against acute promyelocytic Applied Biosystems (Warrington, UK). leukemia (Liu et al., 2012a). Its inhibitory effect on the tumor cell growth and induction of apoptosis can Cell culture be observed in malignant solid tumors as well as acute Human breast cancer MCF-7 cells were obtained from promyelocytic leukemia (Baj et al., 2002; Cheng et al., Pasteur Institute Cell Culture Collection (Tehran, Iran). 2008; Li et al., 2009). Arsenic trioxide induces a paclitaxel- Cells were grown in RPMI 1640 containing 10 % FBS like effect in promotion tubulin polymerization and cell and 100 units/ml penicillin/streptomycin and incubated cycle arrest in most solid tumors (Ling et al., 2002). at 37°C in 5% CO2. Cultured cells at 70-80% confluency Combination of various chemotherapeutic agents can be were washed with pre-warmed phosphate buffered saline an appropriate regimen for overcoming chemoresistance. (PBS) and detached from the flask using 500 µl trypsin- Multiple agents, whether have the same mechanism or EDTA with incubation at 37ºC for 3-5 minutes, followed have antagonistic effects on the cell cycle can participate by addition of culture media containing 10% FBS to with each other in cancer treatment (Ghanbari et al., neutralize the excess trypsin activity. The cell suspension 2014; Samadi et al., 2014). Several studies suggest that was then centrifuged, and the cell pellet was re-suspended combination of multiple agents in comparison with in fresh culture media. single drug, make cancer cells more sensitive (Skidan et al., 2009). Moreover, increasing in the dosage of Development of paclitaxel-resistant MCF-7 cells chemotherapeutic agents could raise their toxicity and side Resistant cells was established as we described effects, therefor, employment of combination therapies can previously (Sharifi et al., 2014). MCF-7 cells with be a useful solution to obtain the maximum effect with the about 20-30% confluency were treated with increasing minimum dosage (Duan et al., 2010; Samadi et al., 2011). concentrations of paclitaxel. Initial treatment concentration Because of widely administration of paclitaxel in was one-tenth of IC50 (0.5 nM) which determined by MTT breast cancer patients, finding new strategies, to overcome assay. Paclitaxel-resistant MCF-7 cells were established chemoresistance will greatly maximize the therapeutic by treating cells with continues and stepwise paclitaxel benefit for individual breast cancer patients. concentrations (0.5 to 64 nM). Culture medium for growth In this study, we investigated the combination effects of MCF-7/PAC resistant cells were enriched with 20% of arsenic trioxide and paclitaxel in paclitaxel-resistant FBS and 10% conditioned medium. Conditioned medium breast cancer cells. We further studied the role of PPM1D was prepared as the supernatant of cultured non-resistant and TP53 in induction of chemoresistance against MCF-7 cells with about 80% confluency. Cells can be a paclitaxel by comparing PPM1D and TP53 expressions in subculture when become confluent then dose of paclitaxel single and combined treatment of resistant cells. Results was increased to 1.5 time of previous dose, and this from our study showed that combination treatment with process continued to reach 64 nM paclitaxel concentration. arsenic trioxide and paclitaxel promotes apoptosis and Generally, cells will tolerate the lower doses of paclitaxel, decreases resistance of the cells to paclitaxel. Down when the cells look not to have tolerated a drug treatment, regulation of PPM1D expression via using arsenic trioxide it is unwise to repeat another round of drug treatment so in combination of paclitaxel, increased the sensitivity of cells maintained in the previous dose for more days. MTT the resistant cells to paclitaxel. Our results suggest that assay was applied to confirm the resistance cells to each combination treatment increases the efficacy of taxans. We dose of paclitaxel. predict that identifying breast cancer patients with high PPM1D expression and then inhibiting this molecule could Assessments of cell viability using MTT assay provide an important adjuvant for improving the efficacy Single Therapy: to determine the drug efficacy in of paclitaxel in these patients. induction of apoptosis after a single exposure, MCF-7/ PAC resistant cells were seeded in 96-well plates with Materials and Methods seeding density of 12,000 cells/well. Then, increasing concentrations of paclitaxel (up to 1000 nM) and arsenic Materials trioxide (up to 9 μM) were applied. Resistant cells were Paclitaxel and arsenic trioxide were purchased from incubated with media containing these agents for 24 and Ebetaxel®, EBEWE Pharma (Unterach- Austuria) and 48 h. Sina Darou (Tehran-Iran), respectively. RPMI-1640 medium and Combined treatment Fetal Bovine Serum (FBS) were obtained from Gibco® To determine the effects of paclitaxel/arsenic trioxide (Invitrogen, USA). 3-(4,5-dimethyl-2- thiazolyl) combinations in cell death, MCF-7/PAC resistant cells -2,5-diphenyl -2H- tetrazolium bromide (MTT),4’,6- were seeded at the density of 12,000 cells/well in 96- diamidino-2phenylindole dihydrochloride (DAPI; 5 well plates. Subsequently, the cells were incubated with mg) and penicillin/streptomycin were obtained from variable concentrations of paclitaxel and arsenic trioxide. Sigma-Aldrich (St Louis, MO, USA). Primers were purchased from Anaspec (Canada). RNX™-Plus Kit MTT Assay and REVERTA-L RT reagent’s kit were obtained The media in each well was replaced with 200 μl 5192 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 DOI:http://dx.doi.org/10.7314/APJCP.2015.16.13.5191 Arsenic Trioxide Promotes Paclitaxel Cytotoxicity in Resistant Breast Cancer Cells fresh media containing 50 μl of MTT. Then the cells were (RT reagents kit). Real time PCR was carried out using incubated for 4 h at 37°C. After incubation period, media/ the SYBR Green-based PCR Master Mix and analyzed MTT mixture was removed and 200 ml of DMSO plus 25 on a Corbett 6000 Rotor-Gene Thermoscycler (Corbett ml of Sorenson’s glycine buffer (0.1 M glycine and0.1 M Research). Total volume of amplification reactions was 25 NaCl, pH 10.5) was added to each well. The absorbance μl, and each well were included 12.5 μl of SYBR Green of each well was measured at 570 nM after shaking for PCR Master Mix, 1 μl of cDNA, 70-100 nM of both 10 minutes, employing a microplate reader (Biotek, ELx forward and reverse special primers. The internal control 800, USA). was the constitutively expressed housekeeping human MTT solution with DMSO (without cells and medium) beta-actin. Primers for human PPM1D were as follows: was used as blank control. sense, 5’-GCC AGA ACT TCC CAA GGA AAG -3’; antisense, 5’-GGT TCA GGT GAC ACC ACA AAT TC Data analysis -3’, for human TP53 were as follows: sense, 5’- TCA ACA Plots of cytotoxicity index (%CI=(1-((OD treaded)/ AGA TGT TTT GCC AAC TG -3’; antisense, 5’- ATG (OD control))×100) versus different concentrations of TGC TGT GAC TGC TTG TAG ATG -3’ and for human each chemotherapeutic agents were drawn. IC50 was beta-actin were as follows: sense, 5’-TGC CCA TCT determined from each plot by calculating the slop and ACG AGG GGT ATG-3’; antisense, 5’-CTC CTT AAT intercept. GTC ACG CAC GAT TTC-3. Samples were measured in The combination index (CI) was calculated using triplicate. Interpretation of the results was performed using the following equation (Eq. 1): CI=(D)1/((D50)1)+(D)2/ the Pfaffle method, and the CT values were normalized (D50)2+α ((D)1 (D)2)/((D50)1 (D50)2) with respect to beta-actin expression.

Where (D50)1 and (D50)2 are the concentrations Statistical analysis of drug 1or 2 alone giving a 50% reduction in cell Results were presented as mean±SD from at least viability compared to a control. (D) 1 and (D) 2 are the three independent experiments. Statistical analysis was concentration of drug 1 and 2 in combination producing a performed using SPSS software through Student’s t-test. A 50% reduction in cell viability compared to a control. a is p-value of less than 0.05 was considered to be statistically 0 when 1 and 2 are mutually exclusive or 1 when they are significant. mutually non-exclusive (Chou and Talalay, 1984). CI <1 was considered synergistic, CI=1 was considered additive Results and CI >1was considered an antagonistic interaction. Anti-proliferatives activity of paclitaxel and arsenic Assessment of apoptotic cells using DAPI staining trioxide on paclitaxel-resistant MCF-7 cells DAPI is known to form fluorescent complexes with The effects of paclitaxel and arsenic trioxide in natural double-stranded DNA. Binding of DAPI to DNA induction of apoptosis on resistant breast cancer cells were enhances its fluorescence strongly. DAPI staining was examined through MTT assay. We first incubated the cells performed as we established previously (Samadi et al., with increasing concentrations of each chemotherapeutic 2009). Cells were seeded in 6-well plates, and after single agent for 24 and 48 h to determine the percentage of and combination treatment for 24 h, cells were fixed with viability for each agent. Paclitaxel was not able to 4 % paraformaldehyde. After 15 minutes, cells were induce 50% cytotoxic effects in resistant cells after 24 washed with phosphate-buffered saline (PBS and then h incubation. Maximum percentage of cytotoxicity was permeabilized with 0.1 % Triton-X-100 for 10 minutes. 44±o.19 %. The IC50 value for paclitaxel after 48 h Cells then were stained with DAPI (1:500 dilution in incubation was 526±0.13 nM. Arsenic trioxide induced PBS) for 10 minutes. Cells were evaluated as normal or 50% cell death in resistant cells with 5.21±0.17 and apoptotic depending on morphological characteristics. 3.4±0.06 μM after 24 and 48 h incubation, respectively Normal nuclei (smooth nuclear) and apoptotic nuclei (p<0.05) (Figure 1a,b). (condensed or fragmented chromatin) were easily distinguished. Triplicate samples were prepared for each Combination treatment restores the sensitivity of resistant treatment and at least 300 cells were counted in random cells to paclitaxel fields for each sample and apoptotic nuclei were identified. To investigate whether incubation of the cells with paclitaxel in combination with arsenic trioxide overcome Assessment of PPM1D and p53 mRNA expression via chemoresistance, the inhibitory effects of different Real-time RT-PCR combinations of paclitaxel/arsenic trioxide were compared Cells were harvested 48 h after incubation with with single therapy. The effect of combination treatment different concentrations of drugs and lysed using lysis on cellular proliferation was evaluated by MTT assay. buffer, RNXPLUS™ (RN7713C) CinnaGen Co., The combined effects of paclitaxel/arsenic trioxide were according to manufacturer protocol. RNA pellet was calculated using isobolographic analysis method. Cells dissolved in DEPC-treated water, quantified by optical were incubated with 250, 500 and 1000 nM of paclitaxel density measurement (A260/A280 ratio) with NanoDrop along with variable concentrations of arsenic trioxide 1000 Spectrophotometer (Wilmington, DE, USA), checked (1-5 µM) for 24 and 48 h. Arsenic trioxide with paclitaxel the quality by agarose gel electrophoresis, and stored at showed an additive or synergistic interaction in MCF-7/ −70°C. cDNA synthesis was done using REVERTAA-L PAC resistant cells after 24h incubation (Table 1).While Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 5193 Tayebeh Oghabi Bakhshaiesh et al paclitaxel was not able to induce 50% cytotoxicity in Combination of paclitaxel and arsenic trioxide promotes resistant cells after 24 h incubation, using combination the formation of apoptotic nuclei in resistant cells treatment increased cytotoxicity of paclitaxel up to To study the effects of single or combination treatment 77.3%±0.01 after 24 h (p<0.05) (Figure 2a). IC50 value for in induction of apoptosis response, cells were stained with paclitaxel decreased to 250±0.21 nM when the cells were DAPI and examined under a fluorescence microscope. incubated with paclitaxel in combination with 4 μM of Minimum 300 cells were examined for each treatment, and arsenic trioxide after 24h incubation (p<0.05). Incubation the percentage of apoptotic nuclei, fragmented nuclei, and of resistant cells with the same combination regimens for condensed chromatin, was calculated. Our results from 48 h, showed mostly synergistic interaction (Table 1). DAPI staining showed that 24 h incubation of the cells After 48 h incubation, 250 nM of paclitaxel along with 3 with 250, 500 and 1000 nM paclitaxel induced 21, 28 and µM arsenic trioxide was able to induced 50% cell death 39% apoptosis, respectively. Furthermore, after incubation in resistant cells (Figure 2b). of resistant cells with 3 and 5 μM of the arsenic trioxide, a population of 29 and 40% apoptotic cells was observed, Table 1. CI Values of Combined Treatment with respectively (p< 0.05) (Figure 3a-c). Incubation of the cells Paclitaxel and Arsenic Trioxide with combined paclitaxel (250, 500 and 1000 nM) and arsenic trioxide (3 μM) for 24 h increased the percentage Drug combination (48h) CI Drug combination (48h) CI of apoptotic cells up to 39, 48 and 56%, respectively PAC* 250+ATO** 1 1.002 PAC 250+ATO 1 1.001 (P < 0.05) (Figure 3d). Our results from DAPI staining PAC 250+ATO 2 1.001 PAC 250+ATO 2 1.001 indicated that combination of two agents could enhance PAC 250+ATO 3 1.002 PAC 250+ATO 3 0.96 the percentage of apoptotic nuclei significantly. PAC 250+ATO 4 0.98 PAC 250+ATO 4 0.89 PAC 250+ATO 5 0.93 PAC 250+ATO 5 0.88 Combination of paclitaxel with arsenic trioxide decreases PAC 500+ATO 1 1.001 PAC 500+ATO 1 0.98 PAC 500+ATO 2 1 PAC 500+ATO 2 0.95 PPM1D expression PAC 500+ATO 3 0.97 PAC 500+ATO 3 0.87 PPM1D, a serine/theronine protein phosphatases, is a PAC 500+ATO 4 0.97 PAC 500+ATO 4 0.82 known negative regulator of cell stress response pathways PAC 500+ATO 5 0.93 PAC 500+ATO 5 0.76 including those regulated by p53 and p38 MAP kinase The combined cytotoxic effect of paclitaxel (250 and 500 nM) with (Lu et al., 2008). As shown in Figure 4a, compared to arsenic trioxide( 1-5 µm) was determined by; CI-isobologram using untreated cells, paclitaxel alone caused a slight decrease in COMPUSYN software; CI > 1, = 1 and < 1 indicate antagonistic, PPM1D expression. Incubation of resistant cells with 250 additive and synergistic effects,respectively; *PAC: Paclitaxel; **ATO: nM paclitaxel along with 3, 4 and 5 µM arsenic trioxide Arsenic trioxide

120 120

100 100

80 80 24 h 24 h 60 48 h 60 48 h

40 40 Cell viability (%) Cell viability Cell viability (%) Cell viability

20 20

0 0 0 50 100 150 200 250 500 750 1000 0 1 2 3 4 5 6 7 8 9 Paclitaxel ( nM ) Arsenic trioxide (µM) Figure 1. Cytotoxic Effects of the Paclitaxel (a) and Arsenic Trioxide (b) in Paclitaxel Resistant Cells After 24 and 48h Incubation. Results are shown as mean ± SD for at least three independent experiments

120 120 PAC* 0 nM PAC 0 nM 100 PAC 250 nM 100 PAC 250 nM Pac 500 nM PAC 500 nM 80 80 PAC 1000 nM PAC 1000 nM

60 60

40 40 Cell viability (%) Cell viability (%) 20 20

0 0 0 1 2 3 4 5 0 1 2 3 4 5 Arsenic trioxide (µM) Arsenic trioxide (µm) Figure 2. Combination Treatment of Resistant MCF-7 Cells with Paclitaxel and Arsenic Trioxide. Treatment of resistant cells with variable concentrations of paclitaxel (PAC) along with arsenic trioxide (1-5 µM) for 24 h (a) and 48 h (b).Data are shown as mean ± SD for at least three independent experiments.*PAC: Paclitaxel 5194 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 DOI:http://dx.doi.org/10.7314/APJCP.2015.16.13.5191 Arsenic Trioxide Promotes Paclitaxel Cytotoxicity in Resistant Breast Cancer Cells A) paclitaxel-only C) treated cells, the TP53 expression was non treated cells increased considerably by combination 3 µM ATO * treatment 5 µM of ATO both agents (Figure 4b).

Discussion

B) Resistance D) to chemotherapeutic agents results in poor 250nM PAC** 500nM PAC 1000nM PAC responses and high 250nM incidence PAC of relapse 250nM in PAC patients with 250nM PAC metastatic breast +3cancer µM ATO (McGrogan et+4 al., µM 2008; ATO Samadi +5 et µM ATO al., 2009). Therefore, understanding about the mechanisms by which cancer cells evade apoptosis and developing new approach to overcome chemo-resistance remain one of the

main obstacles in breast cancer treatment (Melet et al., • v 2008). Using the combination of two or more drugs can be A) C) an effective strategy to overcome resistance by affecting non treated cells 3 µM ATO * 5 µM ATO signaling pathways in cancer cells to promote apoptosis. p53 protein has a central role in response to cellular stress, including DNA damage (Wahl and Carr, 2001; Vousden and Lu, 2002). The leading function of p53 protein as a tumor suppressor is to act as a transcription factor for both

key downstream tumor suppressors and oncogenes (Kong B) D) et al., 2009). Inexistence in p53 function can be the result 250nM PAC** 500nM PAC 1000nM PAC 250nM PAC 250nM PAC 250nM PAC +3 µM ATO +4 µM ATO +5 µM ATO of mutation or depletion of the TP53 gene. On the other hand, tumor cells could use negative feedback to suppress p53 activities (Momand et al., 1998; Bulavin et al., 2002). The aim of this study was to examine whether the combination of paclitaxel and arsenic trioxide can • v suppress resistance to paclitaxel treatment and explore the potential role of PPM1D phosphatase as one of the central Figure 3. Determination of Apoptotic Response in negative regulators of DNA damage signaling pathways. Resistant Cells Upon Treatment of the Cells with In this work, we investigated the anti-proliferatives and Different Concentrations of Agents. DAPI images of: apoptotic effects of paclitaxel (up to 1000 nM) and arsenic a) untreated cells, b) paclitaxel, c) arsenic trioxide and d) trioxide (up to 9 μM) in paclitaxel-resistant MCF-7 cells. combination treated cells have been shown. The results are expressed as mean± SD values for at least three independent Treating resistant cells with 526 nM paclitaxel showed a experiments.*ATO: Arsenic trioxide; **PAC: Paclitaxel percentage of cell death up to 55% after 48 h incubation. The IC50 value for arsenic trioxide after 24 and 48 h 1.2 incubation was 5.21 and 3.4 µM, respectively. These 1 5 results indicated that arsenic trioxide and paclitaxel have 0.8 cytotoxic effect in breast cancer resistant cells at a time- 0.6 and dose-dependent manner, which is consistent with 0.4

0.2 results from previous studies (Liu et al., 2012b). Cells were

mRNA level Relative PPM1D mRNA 0 also incubated with different combinations of paclitaxel control 250 nM PAC* 250 nM PAC+ 3µM250 nMATO** PAC+ 4 250µM nMATO PAC+ 5µM ATO and arsenic trioxide. Treating MCF-7/PAC resistant cells Drug Concentration with the combination of two agents increased cytotoxicity 3.5 by 77.3% after 24 h. While we showed that paclitaxel 3

2.5 was not able to induce 50% cytotoxic effects in resistant 2 cells after 24 h incubation, combination treatment is more 1.5 successful in induction of apoptosis in resistant cells. 1

0.5 In traditional Chinese medicine arsenic trioxide has

mRNA level Relative TP53 mRNA 0 been shown to have anticancer activity, and approved control 250 nM PAC 250 nM PAC+ 3µM250 ATO nM PAC+ 4 µM250 ATO nM PAC+ 5µM ATO by the FDA for the treatment of acute myeloid leukemia Drug concentration (Liu et al., 2012a). The effect of using arsenic trioxide in Figure 4. Determination of a) PPM1D and b) TP53 brain, ovarian, prostate, and some other types of cancers, mRNA Level in Resistant Cells Upon Combination either as a single therapy or in combination with other Treatment with Different Concentration of Paclitaxel anticancer agents, has been implicated (Platanias, 2009; (PAC) and Arsenic Trioxide (ATO). The results are Emadi and Gore, 2010). Various studies suggested that expressed as mean± SD values from at least three independent arsenic trioxide has an activity like paclitaxel, against cell experiments.*PAC: Paclitaxel; **ATO: Arsenic trioxide proliferation in human solid tumors (Ling et al., 2002). Some studies showed that arsenic trioxide produces for 48 h, caused a marked decrease in PPM1D expression. mitotic arrest probably because of targeting tubulin Consistent with this result, compared to untreated and before induction of apoptosis in human breast cancer Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 5195 Tayebeh Oghabi Bakhshaiesh et al cells (Halicka et al., 2002; Cai et al., 2003). Our results comet assay. Mutat Res, 560, 11-7. indicated that arsenic trioxide has a significant impact Bulavin DV, Demidov ON, Saito S, et al (2002). Amplification on the efficacy of paclitaxel in induction of apoptosis of PPM1D in human tumors abrogates p53 tumor-suppressor in resistant cells. The combinatorial treatments led to activity. Nat Genet, 31, 210-5. Burns TF, Fei P, Scata KA, et al (2003). Silencing of the novel elevated apoptosis response and decreased cell viability p53 target gene Snk/Plk2 leads to mitotic catastrophe compared with treatment with paclitaxel alone. in paclitaxel (taxol)-exposed cells. Molec Cell Biol, 23, It has been demonstrated that arsenic trioxide can 5556-71. accumulate apoptosis in some type of cancer cells by Cai X, Yu Y, Huang Y, et al (2003). Arsenic trioxide-induced alteration of the oncogenic Wip1 (Yoda et al., 2008; Meng mitotic arrest and apoptosis in acute promyelocytic leukemia et al., 2011). PP2C serine-threonine phosphatase Wip1 cells. Leukemia, 17, 1333-7. (PPM1D gene) is one of the main negative regulators of Cheng B, Yang X, Han Z, et al (2008). Arsenic trioxide induced the p53 by affecting p53 stability through post translational the apoptosis of laryngeal cancer via down-regulation of modifications or changing Mdm2/p53 interaction (Lu et survivin mRNA. Auris Nasus Larynx, 35, 95-101. Chou TC, Talalay P (1984). Quantitative analysis of dose-effect al., 2005; Lu et al., 2007). Amplification of PPM1D has relationships: the combined effects of multiple drugs or been detected in several different cancers particularly enzyme inhibitors. Adv Enzyme Regul, 22, 27-55. in breast cancer which is usually associated with a poor Dogra S, Bandi S, Viswanathan P, et al (2015). Arsenic prognosis, resistance to radiotherapy and some type of trioxide amplifies cisplatin toxicity in human tubular cells chemotherapies (Fiscella et al., 1997; Parssinen et al., transformed by HPV-16 E6/E7 for further therapeutic 2007; Parssinen et al., 2008; Wang et al., 2012). directions in renal cell carcinoma. Cancer Lett, 356, 953-61. In this study, we examined whether combination of Duan XF, Wu YL, Xu HZ, et al (2010). Synergistic mitosis- arsenic trioxide and paclitaxel can augment p53 expression arresting effects of arsenic trioxide and paclitaxel on human because of down regulation of PPM1D expression. We malignant lymphocytes. Chem Biol Interact, 183, 222-30. Emadi A, Gore SD (2010). Arsenic trioxide - An old drug found that treatment of resistant cells by the combination rediscovered. Blood Rev, 24, 191-9. of two agents decreases the PPM1D mRNA level Fiscella M, Zhang H, Fan S, et al (1997). Wip1, a novel human significantly; while using paclitaxel alone did not show the protein phosphatase that is induced in response to ionizing same effect. We also revealed that combination regimens radiation in a p53-dependent manner. Proc Natl Acad Sci U of these agents could expressively increase the TP53 S A, 94, 6048-53. mRNA level. These results are constant with the previous Ghanbari P, Mohseni M, Tabasinezhad M, et al (2014). Inhibition research, which are reported that arsenic trioxide made of survivin restores the sensitivity of breast cancer cells to a difference in PPM1D expression (Meng et al., 2011; docetaxel and vinblastine. Appl Biochem Biotechnol. Dogra et al., 2015). Goncalves A, Braguer D, Kamath K, et al (2001). Resistance to Taxol in lung cancer cells associated with increased In summary, our data demonstrate that combination microtubule dynamics. Proc Natl Acad Sci U S A, 98, of the arsenic trioxide and paclitaxel is effective in 11737-42. suppressing the tumor proliferation by modulation Wip1/ Gottesman MM (2002). Mechanisms of cancer drug resistance. TP53 expression in paclitaxel-resistant breast cancer Annu Rev Med, 53, 615-27. cells. Arsenic trioxide is more efficient than paclitaxel in Halicka HD, Smolewski P, Darzynkiewicz Z, et al (2002). induction apoptosis in resistant cells. Our data suggest that Arsenic trioxide arrests cells early in mitosis leading to arsenic trioxide can be applied as an adjuant to increase the apoptosis. Cell Cycle, 1, 201-9. efficacy of the therapeutic agents for paclitaxel-resistant Herbst RS, Khuri FR (2003). Mode of action of docetaxel – a breast cancer patients. basis for combination with novel anticancer agents. Cancer Treatment Reviews, 29, 407-15. Ikui AE, Yang CP, Matsumoto T, et al (2005). Low concentrations Acknowledgements of taxol cause mitotic delay followed by premature dissociation of p55CDC from Mad2 and BubR1 and Authors would like to thank Drug Applied Research abrogation of the spindle checkpoint, leading to aneuploidy. Center for supporting this project Cell Cycle, 4, 1385-8. Kavallaris M (1997). The role of multidrug resistance-associated References protein (MRP) expression in multidrug resistance. Anticancer Drugs, 8, 17-25. Ali AY, Farrand L, Kim JY, et al (2012). Molecular determinants Kong W, Jiang X, Mercer WE (2009). Downregulation of of ovarian cancer chemoresistance: new insights into an old Wip-1 phosphatase expression in MCF-7 breast cancer conundrum. Ann NY Acad Sci, 1271, 58-67. cells enhances doxorubicin-induced apoptosis through Baj G, Arnulfo A, Deaglio S, et al (2002). Arsenic trioxide and p53-mediated transcriptional activation of Bax. Cancer Biol breast cancer: analysis of the apoptotic, differentiative and Ther, 8, 555-63. immunomodulatory effects. Breast Cancer Res Treat, 73, Li Y, Qu X, Qu J, et al (2009). Arsenic trioxide induces apoptosis 61-73. and G2/M phase arrest by inducing Cbl to inhibit PI3K/ Bauer JA, Ye F, Marshall CB, et al (2010). RNA interference Akt signaling and thereby regulate p53 activation. Cancer (RNAi) screening approach identifies agents that enhance Letters, 284, 208-15. paclitaxel activity in breast cancer cells. Breast Cancer Ling YH, Jiang JD, Holland JF, et al (2002). Arsenic trioxide Res, 12, 41. produces polymerization of microtubules and mitotic arrest Branham MT, Nadin SB, Vargas-Roig LM, et al (2004). DNA before apoptosis in human tumor cell lines. Mol Pharmacol, damage induced by paclitaxel and DNA repair capability of 62, 529-38. peripheral blood lymphocytes as evaluated by the alkaline Liu H, Tao X, Ma F, et al (2012a). Radiosensitizing effects

5196 Asian Pacific Journal of Cancer Prevention, Vol 16, 2015 DOI:http://dx.doi.org/10.7314/APJCP.2015.16.13.5191 Arsenic Trioxide Promotes Paclitaxel Cytotoxicity in Resistant Breast Cancer Cells of arsenic trioxide on MCF-7 human breast cancer cells cytotoxicity of novel pro-apoptotic agent DM-PIT-1 in PEG- exposed to 89 strontium chloride. Oncol Rep, 28, 1894-902. PE-based micelles alone and in combination with TRAIL. Liu W, Gong Y, Li H, et al (2012b). Arsenic trioxide-induced Drug Deliv, 16, 45-51. growth arrest of breast cancer MCF-7 cells involving Takekawa M, Adachi M, Nakahata A, et al (2000). p53-inducible FOXO3a and IkappaB kinase beta expression and wip1 phosphatase mediates a negative feedback regulation localization. Cancer Biother Radiopharm, 27, 504-12. of p38 MAPK-p53 signaling in response to UV radiation. Lu X, Ma O, Nguyen TA, et al (2007). The Wip1 Phosphatase Embo J, 19, 6517-26. acts as a gatekeeper in the p53-Mdm2 autoregulatory loop. Vousden KH, Lu X (2002). Live or let die: the cell’s response Cancer Cell, 12, 342-54. to p53. Nat Rev Cancer, 2, 594-604. Lu X, Nannenga B, Donehower LA (2005). PPM1D Wahl GM, Carr AM (2001). The evolution of diverse biological dephosphorylates Chk1 and p53 and abrogates cell cycle responses to DNA damage: insights from yeast and p53. Nat checkpoints. Genes Dev, 19, 1162-74. 100.0 Cell Biol, 3, 277-86. 100.0 Lu X, Nguyen TA, Moon SH, et al (2008). The type 2C Wang L, 6.3Mosel AJ, Oakley GG, et al (2012). Deficient DNA 6.312.8 12.8 10.1 20.3 10.1 20.3 phosphatase Wip1: an oncogenic regulator of tumor damage signaling leads to chemoresistance to cisplatin in 10.3 10.3 suppressor and DNA damage response pathways. Cancer oral cancer. Mol Cancer Ther, 11, 2401-9. Metastasis Rev, 27, 123-35. 75.0Yoda A, Toyoshima K, Watanabe Y, et al (2008).25.0 Arsenic trioxide75.0 30.0 25.0 30.0 McGrogan BT, Gilmartin B, Carney DN, et al (2008). Taxanes, augments Chk2/p53-mediated apoptosis by inhibiting 46.8 46.875.0 75.0 microtubules and chemoresistant breast cancer. Biochim oncogenic56.3 Wip1 phosphatase. J Biol Chem, 283, 18969-79. 56.351.1 51.1 Biophys Acta, 1785, 96-132. Zekri A, Ghaffari SH, Yousefi M, et al (2013). Autocrine 51.7 51.7 50.0 54.2 50.0 54.2 Melet A, Song K, Bucur O, et al (2008). Apoptotic pathways human growth hormone increases sensitivity31.3 of mammary 30.0 31.3 30.0 in tumor progression and therapy. Adv Exp Med Biol, 615, carcinoma cell to arsenic trioxide-induced apoptosis. Mol 47-79. Cell Endocrinol, 377, 84-92. Meng XZ, Zheng TS, Chen X, et al (2011). microRNA expression 25.0 25.0 alteration after arsenic trioxide treatment in HepG-2 cells. J 38.0 38.0 Gastroenterol Hepatol, 26, 186-93. 31.3 31.3 30.0 31.333.1 31.3 30.0 33.1 23.7 25.0 23.727.6 25.0 27.6 Momand J, Jung D, Wilczynski S, et al (1998). The MDM2 gene amplification database.Nucleic Acids Res, 26, 3453-9. 0 0 0 0 Parssinen J, Alarmo EL, Karhu R, et al (2008). PPM1D silencing by RNA interference inhibits proliferation and induces apoptosis in breast cancer cell lines with wild-type p53. None None None

Cancer Genet Cytogenet, 182, 33-9. Remission Remission Remission

Parssinen J, Kuukasjarvi T, Karhu R, et al (2007). High-level Radiotherapy Radiotherapy Radiotherapy amplification at 17q23 leads to coordinated overexpression Chemotherapy Chemotherapy Chemotherapy of multiple adjacent genes in breast cancer. Br J Cancer, 96, 1258-64.

Platanias LC (2009). Biological responses to arsenic compounds. recurrence or Persistence recurrence or Persistence recurrence or Persistence J Biol Chem, 284, 18583-7. chemoradiation Concurrent chemoradiation Concurrent chemoradiation Concurrent Rivas MA, Venturutti L, Huang YW, et al (2012). Downregulation of the tumor-suppressor miR-16 via progestin-mediated withtreatment diagnosed Newly withtreatment diagnosed Newly withtreatment diagnosed Newly

oncogenic signaling contributes to breast cancer development. withouttreatment diagnosed Newly withouttreatment diagnosed Newly withouttreatment diagnosed Newly Breast Cancer Res, 14, 77. Rossi M, Demidov ON, Anderson CW, et al (2008). Induction of PPM1D following DNA-damaging treatments through a conserved p53 response element coincides with a shift in the use of transcription initiation sites. Nucleic Acids Res, 36, 7168-80. Sabzichi M, Hamishehkar H, Ramezani F, et al (2014). Luteolin- loaded phytosomes sensitize human breast carcinoma MDA- MB 231 cells to doxorubicin by suppressing Nrf2 mediated signalling. Asian Pac J Cancer Prev, 15, 5311-6. Samadi N, Bekele R, Capatos D, et al (2011). Regulation of lysophosphatidate signaling by autotaxin and lipid phosphate phosphatases with respect to tumor progression, angiogenesis, metastasis and chemo-resistance. Biochimie, 93, 61-70. Samadi N, Gaetano C, Goping IS, et al (2009). Autotaxin protects MCF-7 breast cancer and MDA-MB-435 melanoma cells against Taxol-induced apoptosis. Oncogene, 28, 1028-39. Samadi N, Ghanbari P, Mohseni M, et al (2014). Combination therapy increases the efficacy of docetaxel, vinblastine and tamoxifen in cancer cells. J Cancer Res Ther, 10, 715-21. Sharifi S, Barar J, Hejazi MS, et al (2014). Roles of the Bcl-2/ Bax Ratio, Caspase-8 and 9 in Resistance of Breast Cancer Cells to Paclitaxel. Asian Pac J Cancer Prev, 15, 8617-22. Siegel R, Naishadham D, Jemal A (2013). Cancer statistics, 2013. CA Cancer J Clin, 63, 11-30. Skidan I, Miao B, Thekkedath RV, et al (2009). In vitro

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